Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa

Quorum Sensing allows bacteria to sense their population density via diffusible N-acyl homoserine lactone (N-HL) signaling molecules. Upon reaching a high enough cell density, bacteria will collectively exhibit a phenotype. Until recently, methods used for detection of N-HLs have not considered the chirality of these molecules and it was assumed that only the L-enantiomer was produced by bacteria. The production and effects of D-N-HLs have rarely been studied. In this work, the temporal production of D-N-HLs by the plant pathogen Pectobacterium atrosepticum and the human pathogen Pseudomonas aeruginosa are reported. Both bacteria produced D-N-HLs in significant amounts and in some cases their concentrations were higher than other low abundance L-N-HLs. Previously unreported D-enantiomers of N-3-oxoacyl and N-3-hydroxyacyl homoserine lactones were detected in P. atrosepticum. Interestingly, L-N-HLs produced in the lowest concentrations had relatively higher amounts of their corresponding D-enantiomers. Potential sources of D-N-HLs and their significance are considered.


Introduction
Bacterial cell-to-cell communication occurs via diffusible molecules in a process known as quorum sensing (QS) [1][2][3]. QS involves the biosynthesis of autoinducer molecules and their release outside the cell. Upon reaching a critical concentration, the molecules diffuse back into the cell and are responsible for regulating phenotypic expression. This phenomenon enables bacteria to reach a certain population before exhibiting a phenotype. N-acyl homoserine lactones (N-HLs) are the autoinducer molecules that are responsible for QS in gram-negative bacteria [1,2,4,5]. N-HL molecules consist of a fatty acyl chain attached via an amide bond to a γ-butyrolactone ring, which contains a stereogenic center α to the carbonyl group of the lactone ring [6]. Diversity of N-HLs comes from the varying lengths of the acyl chains and the functionality of the acyl chain. As shown in Fig 1,  To unify varying nomenclature, Table 1 lists the N-HLs explored in this study and their abbreviations that will be used throughout this work. N-HLs indicate all N-acyl homoserine lactones, AHLs represent unsubstituted acyl N-HLs, HHLs represent 3-hydroxy substituted N-HLs and OHLs represent 3-oxo substituted N-HLs. The general notation is D,L-XCY where D,L represents the chirality of the lactone ring, X represents the substitution (A: unsubstituted, H: 3-hydroxyacyl, and O: 3-oxoacyl). Similarly, Y represents the number of carbons on the acyl chain. For HHLs, even though there are two chiral centers, the D,L designation refers to the chirality of the lactone ring. Therefore, there are two L and D diastereomeric pairs respectively.

Chemicals and materials
HPLC grade methanol and acetonitrile, reagent grade dichloromethane, M9 media, D-(+)-glucose, and magnesium sulfate were purchased from MilliporeSigma (St. Louis, MO). A Thermo Scientific™ Barnstead™ GenPure™ Pro water purification system was used to supply deionized (DI) water with 18.

Sample preparation
Standards were diluted to a concentration of 400 μg/mL in acetonitrile from which 1 μg/mL working solutions were prepared for the calibration curve. M9 media was prepared according Table 1. Abbreviations of N-acyl homoserine lactones used in this study.

PLOS ONE
Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa to instructions and autoclaved for sterilization. Then, 20% (w/w) glucose and 1M magnesium sulfate were added to the media. For extraction, an SPE manifold coupled to a vacuum pump was used. The SPE cartridges were conditioned with 10 mL each of acetonitrile, methanol, and water respectively. 10 mL of media was spiked with 7.5 μL of a 4 μg/mL internal standard (D, L-A-C7-N-HL) solution and processed through the SPE cartridge. The column was washed with 10 mL of 95:5 methanol: water and eluted with 11 mL of acetonitrile. The samples were then evaporated using a rotatory evaporator and transferred using dichloromethane. Transferred samples were evaporated using a gentle stream of N 2 , and reconstituted with 100 μL of methanol, then transferred to HPLC vials for analysis. For GC-MS/MS, all samples were prepared similarly with the addition of a derivatization step. The samples were first dried using a gentle stream of nitrogen. Then 75 μL of BSTFA with 1% TMCS and 25 μL acetonitrile (co-solvent) was added to the dried sample, mixed for 10 seconds and eventually placed in a 130˚C sand bath for 45 minutes.

Bacterial samples and growth conditions
Samples for P. aeruginosa ATCC 27853 were obtained from Carolina Biological Supply Company (Burlington, NC). P. atrosepticum SCRI 1043 samples were purchased from American Type Culture Collection (Manassas, VA). Bacterial samples were spiked into M9 media and allowed to grow in a shaker incubator overnight at 30˚C for P. aeruginosa and 25˚C for P. atrosepticum. From the overnight growth, 200 μL of sample was inoculated into 200 mL of fresh M9 media in triplicate. Inoculated media were grown using a shaker incubator at respective temperatures. 11.5 mL aliquots were taken at different time intervals. From the aliquots, 1.5 mL was collected and diluted with 1.5 mL of water and OD 600 reading were taken using a Vernier SpectroVis™ Plus spectrophotometer. The samples were centrifuged for 30 mins and supernatants collected. Supernatants were processed using the SPE method described above and analyzed using LC-MS/MS and GC-MS/MS, for which the conditions are outlined below. Preliminary studies conducted on P. atrosepticum revealed that the production of D-N-HLs was occurring at times >40 h and even up to~120 h. Hence, a 124 h growth period was selected. Similar to P. atrosepticum, preliminary studies of P. aeruginosa were done to select a growth period of 96 h. He was used as a carrier gas with a constant flow of 1.1 mL/min (40 cm/s). The oven temperature was held at 160˚C for 10 minutes and increased at a rate of 1˚C/ min to 230˚C and then held for 50 min. The injection mode was splitless with a 1μL injection and 2 min split time with a 20:1 split ratio. The interface temperature was 230˚C and the MS ion source temperature was 280˚C. Electron ionization (EI) was set to 70 eV. MRM mode was used with optimized collision energies and Q1 and Q3 voltages. All enantiomeric pairs had 2 peaks except for HHLs. HHLs had three peaks for majority of the standard solutions: the first peak is a combination of the two L diastereomers, and the second and third peak represent the D counterparts, which are designated as D1 and D2 respectively (Fig 3). For H-C10 and H-C14, there was some resolution between the L diastereomers. There is ample resolution between homologues for quantitation. All enantiomeric pairs are sufficiently resolved except for O-C6. Peak processing software was used to resolve O-C6 through deconvolution methods and subsequently quantify D-O-C6. The resolution of the analytical signals was obtained by iterative curve fitting using exponentially modified Gaussians. Other peaks were observed but eliminated as matrix peaks based on the retention time and mass transitions.

Quantitation
The calibration curve for LC-MS/MS included concentrations of 5, 12.5, 25, 50, 125, 250, 500, and 1000 ng/mL per enantiomer for all standard N-HLs. The calibration curve for GC-MS/MS included concentrations of 500, 750, 1000, 1500, 2000 ng/mL per enantiomer for all standard N-HLs listed above. Samples for both LC-MS/MS and GC-MS/MS were processed in triplicate using the respective methods outlined above. All quantitation was done using the calibration curves for LC-MS/MS.

Production of D-OHL and D-HHL in P. atrosepticum
The large variety of N-HLs produced by P. atrosepticum during its growth period are summarized in Fig Fig 3. H-C6 N-HL (i.e., a hydroxy-substituted N-HL that has two chiral centers) shows three peaks. The first peak is a combination of the L diastereomers, while the second and third peaks correspond to the D-diastereomers and are designated as D1 and D2 respectively (Fig 3). However, these N-HLs were reported for a different strain of P. atrosepticum. Different strains have been shown to produce distinct N-HLs [10,21,32]. For SCRI 1043, L-O-C6 has been reported as the major N-HL involved in QS [21,32]. This study shows that the D-enantiomer also is produced.   [33,34]. Of these, O-C6 and O-C8 were not detected in this study. Like P. atrosepticum, this could be due to a difference in strain or additionally, the variations in growth conditions. In both bacteria, different classes of N-HLs (AHL, HHL, and OHL) with different chain lengths were detected. Unlike P. atrosepticum, which produced a variety of D-N-HLs, P. aeruginosa only produced D-NHLs with an acyl side chain (i.e., the AHL variety).

Enantiomeric production of N-HLs during the growth of P. aeruginosa and P. atrosepticum
The bacterial growth curve for P. atrosepticum is shown in Fig 5, along with the production of two novel N-HLs, i.e., O-C6 and H-C6. For P. atrosepticum, the exponential growth started at ~12 h, reached a maximum at~36 h and remained constant over the next~100 hours (Fig 5). At its maxima, L-O-C6 had a concentration of 800 ± 300 ng/mL and D-O-C6 had a concentration of 1.01 ± 0.04 ng/mL, while L-H-C6 and D-H-C6 had concentrations of 2.7 ± 0.3 and 0.6 ± 0.3 ng/mL respectively. Two distinct production patterns were observed for L-O-C6 and L-H-C6. The concentration of L-O-C6 followed the OD 600 growth curve until~28 h and

PLOS ONE
Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa declined thereafter (Fig 5A). Detectable amounts of D-O-C6 appeared~24 h and remained constant until the end of the growth period. These patterns were different for L-H-C6 (Fig 5B), which followed the OD 600 curve more closely. D-H-C6 appeared at~56 h and remained constant thereafter. Fig 6 shows the bacterial growth curve of P. aeruginosa along with the production of its more novel N-HLs. Growth started at~9 h, reached a maximum at~25-30 h with the

PLOS ONE
Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa growth decreasing over the next~65 hours (Fig 6). A-C4 and A-C6 were the most produced N-HLs for P. aeruginosa. At the maxima, L-A-C4 and D-A-C4 had concentrations of 600 ±300 and 10±5 ng/mL respectively (Fig 6A). L-A-C6 and D-A-C6 had lower concentrations at 10±4 and 2.0±0.3 ng/mL (Fig 6B). The production of all L-N-HLs in P. aeruginosa followed the same general trend as the growth curve. D-A-C4 also followed the same general pattern as the L-A-C4, i.e, peaking at~25-30 h and then declining slowly until the study was stopped at 96 h. However, the D-H-C6 maintained almost the same concentration (~2 ng/ mL) from~24-96 h.

Comparison of concentrations of L and D N-HLs
The maximum concentrations of N-HLs detected in this study are reported in Table 2. For P. atrosepticum, L-O-C6 was the most produced N-HL, which is consistent with previous findings [21,30]. Most of the L-N-HLs were produced in greater amounts than the highest levels of D-N-HLs. The case of O-C8 is particularly interesting as it was produced in the third highest amount, but its D-enantiomer was undetectable. L-A-C4 and L-H-C6 were produced in lower quantities than O-C8 and had quantifiable levels of their respective D-enantiomers. Interestingly, of the two D-diastereomers in H-C6, only D2-H-C6 was detected. L-A-C4 was the highest produced N-HL for P. aeruginosa, which also is consistent with previous studies [33,34]. Remarkably, D-A-C4 and L-A-C6 were produced in roughly equivalent amounts (both~10 ng/mL) and were the second most prevalent N-HLs. As can be seen in the last column of Table 2, the L/D ratios appear to correlate with the concentration of the L-enantiomer. N-HLs with lower concentrations of L-enantiomers had relatively greater amounts of their D-enantiomers present.

Potential roles and production mechanisms of D-N-HLs in bacteria
D-N-HLs have not been considered to be relevant in QS and have not been reported in bacterial systems until recently in V. fischeri and B. cepacia [9,29]. The methodologies used therein were not sufficiently sensitive to detect D-OHLs and D-HHLs. One of the main goals of this work was to examine the occurrence of D-N-HLs in a broader range of bacteria and specifically, to detect D-OHLs and D-HHLs as shown in Figs 3 & 4. In some cases, the concentration

PLOS ONE
Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa of D-N-HLs were as high as 20% of their L-counterparts. The question arises as to the origin of these D-N-HLs as well as their roles in bacterial systems. P. aeruginosa has two different QS systems involving LasI and RhlI regulons that are activated by the signaling molecules L-O-C12 and L-A-C4 N-HLs respectively [14]. Both of these QS systems are involved in the production of various virulence factors like exoenzymes alkaline protease, and elastase that induces tissue damage in humans [14]. Interestingly, D-A-C6, which doesn't have a defined QS system in P. aeruginosa, was produced in roughly the same concentration as L-O-C12 in this study. Previous investigations on the inhibition and activation of RhlI synthase in P. aeruginosa have revealed that D-A-C4 inhibits this enzyme [35]. In our study, P. aeruginosa produced higher concentrations of D-A-C4 than the LasI signaling molecule L-O-C12. In, P. atrosepticum the highest produced D-N-HL was D-O-C6, which has been shown to activate RhlI [35]. D-O-C6 also has been shown to induce bioluminescence by affecting the LuxR dependent QS system found in V. fischeri [36]. Both P. atrosepticum and P. aeruginosa produced D-A-C6, which has been shown to induce bioluminescence through LuxR as well [36]. Based on these observations, it can be inferred that D-N-HLs can affect the QS systems of their respective L-N-HLs as well as QS systems of other bacteria [35,36]. A study done on cleavage of D,L-O-C12 by the fatty acid amide hydrolase enzyme revealed that the D-enantiomer was relatively resistant to hydrolysis [37]. A different study involving the hydrolysis of the N-HL lactone ring by a lactonase enzyme showed that D-A-C6 was resistant to hydrolysis compared to its L-counterpart [38]. Thus, it is not unexpected that D-N-HLs accumulate with culture time, whereas L-N-HLs eventually decrease, as shown in this study.
There are at least two pathways for the appearance of D-N-HLs: production through a biosynthetic pathway and post-production racemization [9]. D,L-methionine supplementation studies have been conducted to show that production of D-N-HLs through a biosynthetic pathway, utilizing a D-methionine precursor molecule, is unlikely [9]. In terms of post-production, racemization could occur through a racemizing agent or racemization during the sample preparation procedure, and chromatographic analysis. However, auto-racemization during these processes has been ruled out previously [29,39]. Racemization due to pH of the complex biological matrix may be possible, however, analysis of supernatant confirmed that the pH change during the chosen growth periods was insignificant. There is a possibility of an enzyme racemase that may convert the L-N-HLs into D-N-HLs. Such racemases are wellknown for amino acids [40][41][42]. In any case, D-N-HLs are found in lower concentrations than their L-counterparts as in the case of most amino acids [27,43]. Clearly, further investigation is needed in order to understand the origin and role of D-N-HLs in these interesting and important bacterial systems.

Conclusions
Using comprehensive and sensitive LC-MS/MS and GC-MS/MS methods, the presence of D-N-HLs in growth cultures of P. aeruginosa and P. atrosepticum was confirmed. The occurence of unexpected and previously unreported N-HLs was verified for both bacteria. These included novel D-HHLs and D-OHLs which have not been reported previously in any bacteria were. For P. atrosepticum, D-A-C4, D-A-C6, D-O-C6, and D2-H-C6 were found. For P. aeruginosa, D-A-C4 and D-A-C6 were first detected. D-A-C4 was detected in high amounts relative to other L-N-HLs in P. aeruginosa. In general, the concentration of D-N-HLs were usually one to two orders of magnitude lower than their L-counterparts, however, in some cases the ratios were < 10. N-HLs play a role in QS mediated virulence. D-N-HLs have been shown to affect various QS systems. In previous studies, D-A-C4 has been shown to inhibit the L-O-C12 producing QS mechanism involving RhlI while D-O-C6 activated it. D-N-HLs also can be resistant to hydrolysis via fatty acid amide hydrolases and lactonases. Due to these factors, the role of D-N-HLs in bacterial systems should be further explored. If the growing importance of D-amino acids in biological systems is of any indication, the potential role of D-N-HLs in biological systems should be investigated. Of the methods used for analysis, LC-MS/MS was overall more sensitive, and better suited to resolve longer alkyl chain N-HLs. Contrarily, GC-MS/MS gave better resolution of shorter alkyl chain N-HLs, and was used to complement the findings of LC-MS/MS. The origin of D-N-HLs in bacteria is as yet unknown and requires further investigation. As in the case of D-amino acids, a racemase might be involved in the production of D-N-HLs. Likewise, detailed studies on the functions of D-N-HLs in bacterial systems needs to be conducted to explain their potential roles.